The invention relates generally to a method to map a set of biomarker images acquired by a fluorescent microscope into a new color space where the mapped image intensity values represent a brightfield modality.
In traditional histological staining with Hematoxylin & Eosin (H&E), the basophilic dye Hematoxylin (H) is used to stain the cell nuclei blue, and the acidophilic dye Eosin (E) is used as a counter-stain to stain cytoplasm, connective tissue (collagen), muscle fibers, connective tissue, and red blood cells. Eosin interacts with different cellular components in the tissue producing different shades of pink color based on charge properties of the molecules to which eosin are binding. Other chromogenic stains have been used for visualization in immunodiagnostics and assays, for example brown diaminobenzideine (DAB) staining is common.
Often cellular components can be alternatively labeled using molecular markers (dyes and antibodies) with fluorescent dyes. For example, cell nuclei can be stained with DAPI (a fluorescent dye that binds DNA specifically) while other regions in the tissue can be labeled immunofluorescently where the molecules of interest are targeted by directly conjugated antibodies, or by primary secondary amplification detection. For some structures, such as red blood cells (RBC), tissue autofluorescence captured by a set of filters can be used for detection. Fluorescent imaging modality has the advantage of capturing each of these tissue structures individually, hence enabling accurate localization and quantification.
However, histopathological diagnosis based on fluorescent images is not a common practice because fluorescent images do not provide structural and morphological details that are essential for pathologists to diagnose. Brightfield H&E staining, often combined with brown staining techniques, are also often favored as there is a large body of knowledge about these techniques, assembled for decades in pathology laboratories.
Methods to convert fluorescent images into a pseudo brightfield image are known. However, these methods typically reassign a specific color space (wavelength) to each fluorescent dye such that the fluorescent images are recolored into the brightfield space. These methods do not transpose the fluorescent images into an image that represents the image of the biological sample that would be obtained if the biological sample were subjected to a specified brightfield staining protocol, such as H&E. Also disclosed, in U.S. patent application Ser. No. 12/569,396 entitled “Systems and Methods for Generating a Brightfield Image Using Fluorescent Images” and filed on Sep. 29, 2009 is a method which creates a brightfield image from fluorescent images wherein structural features and details of the biological sample are identified as if the image was obtained directly from a specified brightfield staining protocol. The U.S. patent application is incorporated herein by reference.
However a need exist for enhancing contrast and identifying structural features such as cell boundaries. There also exists a need for improving the ability to distinguish and identify internal features such as nucleus, cell membrane and cytoplasm as these areas may lack full resolution. As such a virtual stained image (VSI) with improved acutance, such as edge contrast, is desired.